Expression of chimeric genes in the early region of SV40.

Abstract

Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.